Larger
VersionJay
Groves (foreground) and Pradeep Nair used TIRF imaging and
their own spatial mutation strategy to discover a new way in
which cells can sense and respond to physical forces.

Credit:
Roy Kaltschmidt, Berkeley Lab Public AffairsConventional biological
wisdom holds that living cells interact with their
environment through an elaborate network of chemical signals. As
a result many therapies for the treatment of cancer and other
diseases in which cell behavior goes awry focus on drugs that
block or disrupt harmful chemical signals. Now, a new road for
future therapies may have been opened with scientific evidence
for a never seen before way in which cells can also sense and
respond to physical forces.

A team of researchers with the
Lawrence Berkeley National Laboratory (Berkeley Lab) and the
University of California (UC) Berkeley has shown that the
biochemical activity of a cellular protein system, which plays a
key role in cancer metastasis, can be altered by the application
of a direct physical force. This discovery sheds important new
light on how the protein signaling complex known as
EphA2/ephrin-A1 contributes to the initiation, growth and
progression of cancerous cells, and also suggests how the
activity of cancer cells can be affected by surrounding tissue.

“This first evidence that
the EphA2/ephrin-A1 receptor-ligand complex, which was previously
thought to be strictly a chemical sensor, can actually sense
mechanical properties as well,” says chemist Jay Groves,
who led this research. “This coupling of mechanical and
chemical signaling, which could never have been seen with
classical biological methods, helps explain some of the
biological mysteries concerning the onset and progression of
cancer.”

Groves holds a joint
appointment with Berkeley Lab’s Physical Biosciences
Division and UC Berkeley’s Chemistry Department. He is also
a Howard Hughes Medical Institute (HHMI) investigator. With
members of his research group Khalid Salaita and Pradeep Nair,
plus Rebecca Petit, he has co-authored a paper on this research
that was published in the March 12, 2010 issue of the journal
Science. The
paper is titled, “Restriction of Receptor Movement Alters
Cellular Response: Physical Force Sensing by EphA2.” Other
co-authors were Joe Gray, Richard Neve and Debopriya Das of
Berkeley Lab’s Life Sciences Division.

Cancer
and EphA2/ephrin-A1

The term “metastasis”
comes from the Greek word for “displacement,” and it
is used to describe the process whereby cancer cells detach from
a tumor, enter the bloodstream and spread to other tissues
throughout the body. For example, cancerous breast cells can
spread to a lung and form a new breast cancer tumor there.
Central to metastasis is the EphA2/ephrin-A1 receptor-ligand
complex.

EphA2 is a member of the
receptor tyrosine kinase (RTK) family of enzymes that are key
regulators of cellular processes. The over-expression of EphA2
has been linked to a number of human cancers, including melanoma,
lung, colon and prostate, but is especially prominent in breast
cancer. Some 40-percent of all breast cancer patients show an
over-abundance of EphA2, with the highest levels found in the
most aggressive cancer cells. Ephrin-A1 is a signaling protein
that is tethered to the surface of a cell’s outer membrane.
It binds to EphA2 in a neighboring cell like a key fitted into a
lock. When ephrin-A1 binds with EphA2, the newly bound complexes
become activated and gather in a cluster.

“The host cell will then
literally give the clusters a distinctive tug, applying a force
that pulls the clusters across the surface of the cell to a
centralized location,” Grove says. “What we found is
that by applying an opposing force, we could alter the cell’s
biochemical activity. When we applied a big opposing force we
were able to convert highly invasive cells into well-behaved
cells. This shows that in addition to chemically sensing the
presence of ephrin-A1, the cells also sense the mechanical
properties of the local environment in which ephrin-A1 is
displayed.”

Spatial
Mutation

Larger
VersionMetal
lines patterned into a silica membrane beneath a cell act as
a diffusion barrier, impeding the mobility of EphA2/ephrin-A1
signaling complexes so they accumulate along the boundaries
of the barrier. Without the barrier, the complexes are
transported to a centralized location within the cell.

Credit:
Berkeley LabObservations have indicated
that mammalian cells are sensitive to the physical aspects of
their environment, such as the texture or geometry of the
surrounding tissue. However, evidence that physical forces impact
freely-moving signaling molecules (as opposed to focal
adhesion molecules) in the membranes of cells has been lacking
because the cell membrane is an environment that has always been
difficult to characterize and manipulate. Groves and his research
group have found a way to overcome this obstacle with the
development of unique synthetic membranes constructed out of
lipids and assembled onto a substrate of solid silica that
enables them to directly control cellular signaling activities.

“We call this approach
the ‘spatial mutation’ strategy because molecules in
a cell can be spatially re-arranged without altering the cell in
any other way,” Groves says. “We first used this
strategy in 2005 to study T cell signaling in the immune system.”

In this latest study, Groves
and his colleagues worked with mammary epithelial cells from a
library of 26 model human breast cancer cell lines that have been
well-characterized by co-author Gray and his research groups at
Berkeley Lab and UC San Francisco.

Says co-author Nair, “Gray’s
research has demonstrated that this library substantially
reproduces the genomic abnormalities and drug responsiveness of
primary breast cancer tumor cells from patients, and constitutes
the most comprehensive system for the study of the various
aberrations responsible for human breast cancer.”

To test the sensitivity of the
EphA2/ephrin-A1 signaling complex to mechanical forces, Groves
and his group patterned their silica substrates with chromium
metal lines that were 10 nanometers in height and 100 nanometers
wide. These metal lines acted as diffusion barriers that impeded
the lateral mobility of the EphA2/ephrin-A1 complexes in the
synthetic membrane. The movement and spatial organization of the
complexes were subsequently tracked through a combination of
Total Internal Reflection Fluorescence (TIRF), reflection
interference and epifluorescence imaging techniques.

“Without the barriers,
the clusters of EphA2/ephrin-A1 signaling complexes were
transported to the center of the cell–supported membrane
junction, but with the barriers in place, there was an
accumulation of clusters at the barrier boundaries,” Groves
says. “This resulted in a spatial reorganization that
altered the cell’s biochemical behavior.”

Quantitative analysis of these
changes to the spatial organization of the EphA2/ephrin-A1
signaling complexes across the library of breast cancer cell
lines revealed a strong correlation with the potential for
metastasis. Since the patterned metal lines in the silica
substrate are analogous to the stiffness, texture and other
elastic and mechanical properties of tissue, as well as to
internal structures within the cell membrane, the results of this
study point to intriguing new possibilities for breast and other
cancer therapies.

“It’s possible that
the force-sensing process itself could provide a target for
therapeutic intervention,” says Groves. “We’re
also excited about finding targets for which there may be drugs
that have already been developed but are now being used to treat
diseases other than cancer. Given the sensitivity to mechanical
forces displayed by the EphA2/ephrin-A1 signaling complexes, it
is possible these existing drugs could be redirected to the
treatment of cancer.”

This research was carried out
under a grant from the Bay Area Physical Sciences-Oncology
Center, which is funded by the National Cancer Institute to
enable the tools and insights of the physical sciences to be
applied to the investigation of cancer’s underlying
mechanisms. This center is under the direction of Jan Liphardt, a
biophysicist who also holds joint appointments with Berkeley Lab
and UC Berkeley.

Berkeley Lab is a U.S.
Department of Energy national laboratory located in Berkeley,
California. It conducts unclassified scientific research and is
managed by the University of California.